1. Field of the Invention
The present invention relates to a decaprenyl diphosphate synthetase, a gene coding for the synthetase, a recombinant vector comprising the gene, a transformant transformed with the vector, a method for producing a decaprenyl diphosphate synthetase, and a method for producing ubiquinone-10.
2. Description of the Prior Art
Isoprenoids are the most varied group of compounds including more than 23,000 species occurring in nature. They include sterols, carotenoids, sugar carrier lipids, prenyl quinones, prenylated proteins, etc. (FIG. 1). Those enzymes which catalyze the formation of carbon skeletons that will be the basis for the biosynthesis of these isoprenoid compounds (i.e., enzymes which catalyze the head-to-tail type condensation polymerization of isopentenyl diphosphate (IPP) that is 5-carbon isoprene unit) are generically called as prenyl diphosphate synthetase. Prenyl diphosphate synthetase is classified into 4 groups depeding on the chain length, conformation, etc. of the prenyl diphosphate generated (Table 1).
Short-chain prenyl diphosphate synthetase (prenyltransferase I) includes geranyl diphosphate (GPP, C10) synthetase, farnesyl diphosphate (FPP, C15) synthetase (Eberhardt, N. L. et al., (1975) J. Biol. Chem. 250:863-866), geranylgeranyl diphosphate (GGPP, C20) synthetase (Sagami, H. et al. (1994) J. Biol. Chem. 269: 20561-20566) and the like. The short-chain prenyl diphosphates biosynthesized by these enzymes are water-soluble. They may be supplied as an allyl primer substrate for polyprenyl diphosphate synthetase belonging to other groups.
Medium-chain prenyl diphosphate synthetase (prenyltransferase II) includes hexaprenyl diphosphate (HexPP, C30) synthetase (Fujii, H. et al., (1982) J. Biol. Chem., 257:14610), heptaprenyl diphosphate (HepPP, C35) synthetase (Takahashi, I. et al., (1980) J. Biol Chem., 255: 4539) and the like. These enzymes are greatly different from the short-chain prenyl diphosphate synthetase described above in that they are heterodimeric enzymes composed of two proteins each of which does not have a catalytic function alone. Usually, these two proteins are dissociated, but when a substrate is present, they associate with each other to manifest a function as an enzyme. Although those products produced by such enzymes are highly hydrophobic and apt to form micelles, they do not require lipids nor surfactants for the manifestation of their enzyme activity. This is considered due to the fact that the medium-chain prenyl diphosphate synthetase is a special system in which such dynamic dissociation and association are repeated.
E-type long-chain prenyl diphosphate synthetase (prenyltransferase III) includes octaprenyl diphosphate (OctPP, C40) synthetase, decaprenyl diphosphate (DPP, C50) synthetase and the like. Unlike prenyltransferase II, these enzymes are undissociable homodimers and activated by polyprenyl diphosphate carrier proteins (Ohnuma, S. et al., (1991) J. Biol. Chem. 266: 23706-23713). This activation is believed to maintain the catalyst turnover by removing hydrophobic reaction products from the active sites of these enzymes.
Z-type long-chain prenyl diphosphate synthetase (prenyltransferase IV) includes nonaprenyl diphosphate (E,E-farnesyl-all-Z-hexaprenyl diphosphate, C45) synthetase, undecaprenyl diphosphate (E,E-farnesyl-all-Z-octaprenyl diphosphate, C55) synthetase and the like. Reaction products generated by these enzymes work as sugar carrier lipids in the biosynthesis of bacterial cell walls. These enzymes need the addition of a phospholipid or surfactant for the manifestation of their activity. DPP synthetase, which is classified into prenyltransferase III, is also known to require a surfactant for the manifestation of its enzyme activity.
A soil bacterium Paracoccus denitrificans is a bacterium which is believed to be the origin of human mitochondria. The respiratory chain and the oxidative phosphorylation mechanism of this bacterium are more efficient and more united as one organization than those of other bacteria. Thus, the characteristics of P. denitrificans are more closer to those of mitochondria (John, P. et al., (1975) Nature, 254, 495-498). Three types of prenyl diphosphate synthetase activities have been confirmed from P. denitrificans (FIG. 2). They are activities of (i) FPP synthetase which catalyzes E-type condensation of dimethylallyl diphosphate (DMAPP) with 2 molecules of IPP to produce FPP; (ii) nonaprenyl diphosphate (NPP) synthetase which catalyzes Z-type condensation of FPP with 6 molecules of IPP to produce NPP (Ishii, K. et al., (1986) Biochem. J., 233, 773-777); and (iii) DPP synthetase which catalyzes E-type condensation of FPP with 7 molecules of IPP to produce DPP (Ishii K. et al., (1983) Biochem. Biophys. Res. Commun., 116, 500-506).
NPP produced by NPP synthetase becomes a sugar carrier lipid which is essential for the biosynthesis of the cell wall of this bacterium. However, unlike several E-type prenyl diphosphate synthetases which have been already cloned and analyzed, prenyl diphosphate synthetases such as NPP synthetase and undecaprenyl diphosphate synthetase which catalyze Z-type condensation reaction have not been elucidated yet in relationships between their structures and enzymatic functions.
DPP produced by DPP synthetase is metabolized on the prenyl side chain of ubiquinone-10 (a constituent of the electron transport system) produced by this bacterium. All of the C30-C50 polyprenyl diphosphates biosynthesized by bacterial prenyltransferase II or III are provided as a side chain precursor of the corresponding menaquinone or ubiquinone. Therefore, the chain length of the product of each enzyme is directly reflected in the side chain length of the prenylquinone of the bacterium from which the enzyme is derived. Among prenylquinones, ubiquinone-10 is industrially extracted from Paracoccus denitrificans and used as pharmaceuticals since it has the same side chain length as that of human coenzyme Q (CoQ). Ubiquinone has been known to be effective for chronic heart diseases (Yamamura, T. (1977) Clinical Status of Coenzyme Q and Prospects 281-298). Ubiquinone-10 is also effective as an antiarrhythmic agent and, thus, is utilized for the prevention of arrhythmia and the like (Fujioka, T. et al. (1983) Tohoku J. Exp. Med. 141, 453-463).
It is the object of the present invention to provide a decaprenyl diphosphate synthetase, a gene coding for the synthetase, a recombinant vector comprising the gene, a transformant transformed with the vector, a method for producing the decaprenyl diphosphate synthetase, and a method for producing ubiquinone-10.
As a result of intensive and extensive researches toward the solution of the above assignment, the present inventor has succeeded in cloning a gene coding for a long-chain decaprenyl diphosphate synthetase from Paracoccus denitrificans. Thus, the present invention has been achieved.
The present invention relates to a recombinant protein (a) or (b) described below:
(a) a protein having the amino acid sequence shown in SEQ ID NO: 2
(b) a protein which has the amino acid sequence shown in SEQ ID NO:2 having deletion, substitution or addition of at least one amino acid and which has decaprenyl diphosphate synthetase activity.
The present invention also relates to a gene coding for the recombinant protein (a) or (b) described above. Specific examples of this gene include a gene comprising the base sequence shown in SEQ ID NO: 1.
Further, the present invention relates to a recombinant vector comprising the above gene.
The present invention further relates to a transformant transformed with the above vector.
The present invention further relates to a method for producing a decaprenyl diphosphate synthetase comprising culturing the above transformant in a medium and recovering a decaprenyl diphosphate synthetase from the resultant culture.
The present invention further relates to a method for producing ubiquinone-10 comprising extracting ubiquinone-10 from the above transformant.